Method of sanitizing edible seeds, particularly mucilage producing seeds

ABSTRACT

Edible seeds are sanitized with a composition comprising water, a biocidal agent and a solvent. The composition is applied to the seeds and the seeds are later dried without first rinsing the seeds. The application rate may be 15% by weight of the seeds or less. The solvent may be a water-miscible alcohol such as ethanol or propylene glycol. When sanitizing mucilaginous seeds, the composition comprises one or more alcohols in an amount effective to inhibit the release of mucilage. The composition is initially applied as a mist to mucilaginous seeds. The biocidal agent may be an oxidizer such as peracetic acid. A treatment system includes an atomizing sprayer and a dryer, for example a fixed bed forced-air dryer.

RELATED APPLICATIONS

This application claims priority from, and for the U.S.A. is also acontinuation-in-part of, International Application NumberPCT/CA2014/051088, A Novel Composition and Method of Use to ControlPathogens and Prevent Diseases in Seeds, filed on Nov. 13, 2014 byAgri-Neo Inc., which is incorporated by reference.

FIELD OF THE INVENTION

The invention relates to the preservation of edible seeds, includingsprouted seeds.

BACKGROUND

Seeds, such as flax, chia, hemp and sesame seeds, are economicallyvaluable and nutritious food products. However, there are many potentialpoints of entry for pathogens in the seed processing chain betweenharvesting and packaging. Some pasteurized seeds are commerciallyavailable. However, pasteurizing cooks the seeds and is not an optionfor seeds that are marketed as raw. The seeds are believed to have morenutritional value when raw, and raw seeds are at preferred by at leastsome consumers.

Some raw fruits and vegetables have been sanitized with aqueouscompositions. For example, U.S. Pat. No. 2,512,640 describes the use ofperacetic acid (also known under the tradename Peracid) for thetreatment of raw fruits and vegetables to reduce spoilage from bacteriaand fungi before processing. Peracetic aqueous solutions have also beensuggested to control pathogenic organisms on growing plants(International Patent Publication WO 2012/051699 and U.S. Pat. Nos.6,024,986; 6,165,483; and, 6,238,685).

As a consequence of their small size, however, seeds have a much largersurface area for a given volume than most fruits and vegetables. Aqueoussanitizing solutions generally employ a contact killing mechanism and soefficacy depends on coverage. This suggests that a large volume of anyaqueous composition would be required to treat seeds, but seeds aretypically stored and processed dry.

Some seeds also release mucilage when wet. Mucilage is a polysaccharidewith a high swelling index that produces a viscous solution in water.Mucilaginous seeds contain mucilage-secreting cells (MSCs) primarilylocated in the seed coat, or epidermal layer of the seed. When the seedsare dry, the mucilage is contained in cell wall structures, for examplebetween primary and secondary cells walls. When the seeds contact water,the mucilage swells, breaks free of the cell structures, and covers theseeds with mucilage. The mucilage is edible but, if secreted, it bindsthe seeds together making the seeds difficult to process and store.Examples of commercially important mucilage producing seeds include flaxand chia.

Current food safety practices for raw seeds rely on sampling. Whensampling detects an excess of pathogens, large containers of seed arewasted. And yet sampling also fails to detect all contaminatedshipments. For example, sesame, chia and flax seeds have all causedoutbreaks of Salmonella poisoning among people eating the raw seeds.

INTRODUCTION TO THE INVENTION

This specification describes a system and method for sanitizing rawedible seeds with an aqueous composition. The system and method can beused to provide preventative sanitization, or to sanitize seeds thatalready have an unacceptable pathogen concentration. The seeds may bewhole, or in alternative forms such as sprouted, cracked or powdered.

The method involves applying an aqueous sanitizing composition to theseeds. The composition includes at least one biocidal agent and at leastone solvent. Preferably, the solvent is a water-miscible alcohol that isfood-grade, volatile, or both. Some examples of suitable solventsinclude ethanol and propylene glycol. The biocidal agent may be anoxidizer such as peracetic acid.

The composition is applied sparingly to the surface of the seeds. Forexample, the application rate may be 15% by weight of the seeds or less.Sufficient sanitizing may occur essentially on initial contact, orwithin about 5 minutes, but contact time may be extended to furtherreduce the number of living pathogens on the seeds. However, the seedsare dried no more than 48 hours, preferably no more than 24 hours, afterapplying the composition. Drying returns the seeds to a moisture contentsuitable for storing the seeds, and preferably vaporizes at least mostof the solvent and optionally the biocidal agent. The seeds are notrinsed before they are dried. The seeds can be dried, for example, byblowing air over them.

The method may be used for sanitizing mucilaginous seeds, such as chiaand flax seeds. In this case, the aqueous sanitizing composition isprepared with an alcohol, or a mixture of alcohols, in an amounteffective to suppress mucilage release from the seeds. For example, thecomposition may comprise at least 15% v/v ethanol, or at least 13% v/vpropylene glycol. The composition is initially applied to themucilaginous seeds as a mist. The composition sanitizes the seedswithout causing mucilage production.

The system includes equipment adapted to perform the method. Preferredapplication equipment has one or more atomizing sprayers, optionallywith a downstream mixer. Preferred drying equipment includes a fixed beddryer with forced-air flow through the bed.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 to 3 represent results obtained according to Example 2.

FIGS. 4 to 6 represent results obtained according to Example 3.

FIG. 7 is a photograph of an application device having a sprayer, ahopper and an auger.

FIG. 8 is a graph of experimental results showing aerobic counts atvarious incubation times.

DETAILED DESCRIPTION

Popular raw edible seeds include hemp, flax, sesame and chia seeds. Rawseeds can be eaten whole, sprouted, cracked or as a powder. Thedescription below describes an aqueous sanitizer composition, which maybe referred to as a sanitizer or composition for brevity. The sanitizercan be used to kill disease-causing pathogens such as viruses, bacteria,fungi, yeasts and molds. Common bacterial pathogens include Salmonella,Listeria monocytogenes and E. coli. The sanitizer may be used, forexample, to control E. coli in hemp seeds and Salmonella in chia seeds.The sanitizer is typically used after the seeds have been harvested andbefore they are packaged. The sanitizer may be used as a preventativetreatment, or after an unacceptable contamination has been detected.

Seeds are difficult to treat with an aqueous sanitizer compositionbecause they are small, and have a very high surface area per unitvolume. Since seeds are also generally stored and processed in a drystate, it is preferable not to apply a large volume of the sanitizer.Some seeds also release mucilage when they contact water. In the methoddescribed below, an aqueous sanitizer includes one or more biocidalagents and one or more solvents. The solvent is believed to helpdisperse the composition across the seed. Applying the composition as amist also helps disperse the compositions, and is particularly usefulfor treating mucilaginous seeds. The composition can be effective evenat low application rates, for example 15 wt % (150 L/tonne) or less or 5wt % (50 L/tonne) or less. After applying the composition, the seeds aredried typically to or near their original moisture content, for examplewithin 1% by weight of the seeds of their original moisture content.This inhibits regrowth of microorganisms and, preferably, vaporizes thesolvent and possibly the biocidal agent. The seeds do not need to berinsed of the composition.

The term “solvent” is used as in the chemical engineering vernacular tomean an organic liquid capable of dissolving a variety of compounds. Thesolvent should be miscible in water, at least to some concentration. Thesolvent is preferably volatile or food grade or both. Ideally, thesolvent should be generally regarded as safe (GRAS) according to anyrelevant law dealing with the production or sale of food in the relevantjurisdiction. To enable larger amounts of solvent to be used, thesolvent may be a hydroxylated hydrocarbon, i.e. an alcohol. Examples ofuseful solvents include alcohols such as ethanol and propylene glycol.Other potentially useful solvents include glycol ethers, ethyleneglycol, isopropanol, and monobutyl ether of ethylene glycol. The solventmay represent from 2-70% by volume, preferably 50% or less by volume,optionally less than 20% by volume, but preferably at least 15% byvolume, of the total volume of the composition.

In general, lower alcohols are useful solvents because of theirmiscibility in water. For example, the solvent may be selected from thegroup consisting of C₁-C₆ alcohols and glycol ethers. In particular, thesolvent may be an alcohol of formula ROH where R represents a linearalkyl group having from 1 to 6 carbon atoms, or a branched alkyl grouphaving from 3 to 6 carbon atoms. The alcohol may also be a food-gradealcohol that is listed in FDA's CFR 21 as Generally Regarded as Safe(GRAS) for use in food (section 184.1293), such as ethanol, propanol orisopropanol.

Alcohol solvents, and possibly some others, also allow the compositionto be applied to mucilaginous seeds without causing them to expressmaterial amounts of mucilage. The reason why alcohols suppress mucilagerelease when added to the composition is unknown. Without intending tobe limited by theory, the inventors believe that the effect may be aresult of a change in polarity of the composition relative to water.Alternatively, the effect may be related to the way that an alcoholcauses DNA, for example, to precipitate out of solution with water,although the amount of alcohol required to inhibit mucilage release ismuch lower.

The effective amount required to suppress mucilage release variesslightly between different alcohols, but can be easily determined bytrials at different concentrations. The effective amount for ethanol isa concentration of about 15% by volume. The effective amount forpropylene glycol is a concentration of about 13% by volume. Theeffective amount of a 50:50 mixture of propylene glycol and ethanol is aconcentration of about 14% by volume. Effective amounts for other mixesof ethanol and propylene glycol can be obtained by linear interpolationof theses results. Alternatively, since ethanol is a light, low alcoholyet the effective amount for propylene glycol (a diol of higher density)is not markedly different, these results suggest that about 15% byvolume is likely to be effective for any alcohol or mixture of alcohols.The effective amount appears to be related to the concentration of thealcohol independent of the application rate of the alcohol.

Higher solvent concentrations may also be used. Concentrations of up to50% ethanol have been tested and found effective. Ethanol at a 15%concentration is generally biostatic, it does not cause significantgrowth or death of most pathogens. Ethanol at 50% concentrationincreases the biocidal effect of the sanitizing composition. However,the increased biocidal effect is most significant with short contacttimes, for example about 3 to 12 hours between initial application anddrying the seeds. With longer contact times, the increase in biocidaleffect attributable to the ethanol decreases. For example, in tests onhemp seeds an increase in contact time from 12 hours to 24 hours wasfound to be more effective than adding 50% ethanol, both cases beingrelative to a formulation without ethanol applied for 12 hours.Depending on the practical circumstances of any particular plant, thisobservation may indicate (a) that efficacy can be obtained withoutincreasing solvent concentration by extending the contact time or (b)that efficacy can be obtained with a short contact time by increasingsolvent concentration.

Some solvents are also difficult to handle at high concentration. Forexample, an aqueous mixture containing ethanol at more than 20% byvolume is considered flammable while a mixture with 20% or less ethanolis considered combustible. Combustible materials are safer and havefewer handling requirements. Accordingly, a solvent concentration of 20%by volume or less may be preferred in some cases. An ordinary mixer canbe used to prepare sanitizer compositions with 20% or less ethanol.

Even with a solvent, treatment of mucilaginous seeds in particular issensitive to the initial application of the composition. It ispreferable to disperse the composition and provide a large initialcontact area rather than to rely on mixing after initial application todisperse the composition. For example, applying the composition throughan atomizer to produce a mist improves the efficacy of the treatmentover applying the composition in a continuous spray. The composition canbe applied as an atomized spray to the seeds or the seeds can fallthough a mist of the composition. In one example in which a continuousspray was used to initially apply a composition to chia seeds, mucilagedid not form but the microbial kill was not effective. However, the chiaseeds were effectively sanitized when a similar composition was appliedwith an atomized spray.

Agricultural seed treatment equipment, normally used to apply anantimicrobial or fungicidal chemical to seeds before planting them, ormodifications thereof, can be used. For non-mucilaginous seeds, theinitial application is less critical and an ordinary sprayer may beused, followed by secondary mixing. Secondary mixing is also useful evenwhen the composition is applied as a mist and can be performed, forexample, in an auger, tumbler or rotating drum. Agricultural seedtreating equipment may provide both initial application and secondarymixing. Suitable equipment includes the USC Continuous Treating System,Bayer RH series treaters, USC LPX series treaters and the KSi 4808NGAapplicator.

The biocidal agent in the composition may be any one or more biocidalagents in amounts effective to sanitize seeds to a level safe for humanconsumption. Suitable oxidants include peracetic acid, hydrogenperoxide, iodine, chlorine, bromine and chlorine dioxide. A mixture oftwo or more oxidants may also be used. The one or more oxidants may bepresent in a weight ratio ranging from 1:100 to 1:4, for example 1:20 to1:5, relative to the water. Optionally, the composition may include asurfactant.

Peracetic acid (C₂H₄O₃) is particularly useful because it effectivelykills on contact and essentially vaporizes when the seeds are dried.Paracetic acid can be obtained as a liquid preformed product orgenerated in-situ from powder precursors. Peracetic acid in an aqueoussolution is a mixture comprising acetic acid (CH₃COOH) and hydrogenperoxide (H₂O₂). Typically, peracetic acid (also identified hereinafterunder the acronym PAA) is produced by reacting acetic acid and hydrogenperoxide. Methods of generating a liquid solution comprising PAAstarting from the dissolution of a powdered mixture are described inU.S. Pat. No. 7,291,276; UK patent application No. 2,355,198; FR patentapplication 2,728,171; Canadian patent application No. 2,569,025;International PCT patent application WO 95/02330 and EP patentapplication No. 0 648 418, which are incorporated by reference.

The treatment process, in summary, includes providing a sanitizingcomposition having a solvent and a biocidal agent mixed with water. Thiscomposition is applied to the seeds. The application rate is preferablyless than 15% by weight of the seeds, typically 3-5% (30-50 L/tonne).Optionally, there may be secondary mixing after the initial application.Sufficient sanitizing may occur essentially on initial application, orin the time taken for secondary mixing and transport to a dryer, forexample in about 5 minutes. Optionally, the seeds may be stored afterinitial application for an hour or more with the composition in contactwith the seeds. The seeds are dried to end the process. Drying continuesuntil the seeds return to or near their starting moisture content, or atleast until the seeds are below a moisture content, for example 10 wt %,suitable for storing them. The seeds do not need to be rinsed beforethey are dried, and mucilaginous seeds in particular are not rinsed.

To store the seeds in contact with the composition, the seeds can beconveyed into solid (non-porous) storage bins. The bins are preferablycovered, but not airtight. Some of the solvent may evaporate during thecontact time, and it is preferable to allow the solvent to escape toavoid having a flammable gas over the seeds. In one trial, seeds weretreated with a 50% ethanol composition and stored in a conventionalcovered agricultural grain bin. There was no detectible ethanol in airin the bin when measured 16 hours after adding the seeds.

The seeds can be dried in a fixed or moving bed by sucking or blowinghot air through the bed. The air is heated, primarily to increase theability of the air to hold water. This allows the air to dry the seedseven if the air is initially saturated. Alternatively or additionally,the air may be dehumidified before it passes through the bed of seeds.However, the air should not be heated to an extent that would render theseeds no longer raw. Different standards exist for marketing raw seeds,and the maximum temperature may be in the range of 37-70° C., typically40-49° C. For example, the air may be heated to not more than 40° C. Theseeds can be dried in agricultural grain driers or in fixed bed batchdriers. Fixed bed batch driers are preferred since they produce lessdust and seed damage. The drier can be, for example, a circular bindryer with air supplied to a vertically oriented central porous tubeinside of a cylindrical bin holding the seeds. The cylindrical bin has asolid bottom and porous sides. A floating solid cover can be used toallow for the bed to contract as it dries. Optionally, an outside-inairflow path may be used to provide increased air velocity across thedownstream seeds. In another option, frusto-conical central tube orouter bin walls can be used to provide more nearly equal airflow throughthe top and bottom of the bed. Alternatively, commercial rectangulardrying boxes can be used, optionally to both hold the seeds during acontact time and while drying the seeds.

Example #1 Protocols for Seeds, Drains and Spices SanitationI—Preparation of Different Solutions

1. Wetting Agent (i.e. Surfactant) Preparation:

Description:

-   -   APG® 325 is a liquid wetting agent (i.e. a surfactant) composed        of alkyl polyglycoside and derived from natural sources. It is a        foaming surfactant.

Preparation:

-   -   5 g of liquid APG 325 surfactant were diluted in 1 L water, and        mixed for 5 minutes, to make 0.5% solution of wetting agent        (i.e. surfactant).

2. Alcohol Preparation:

Description:

-   -   A food-grade alcohol based on ethanol at 94% concentration        minimum (provided by Greenfield Ethanol).

Preparation:

-   -   100 ml of the above-mentioned ethanol were diluted in 100 ml        water to a make a 50% food grade ethanol.

3. Powdered Peracetic Acid Preparation without Surfactant:

Description:

-   -   A blend of sodium percarbonate (62% w/w), TAED (20% w/w) and        citric acid (18% w/w) that generates peracetic acid and hydrogen        peroxide in-situ. It is a non-foaming solution and free of        surface-active agents. This Powdered PAA is equivalent to 10%        peracetic acid.

Preparation:

-   -   100 g of Powdered PAA was dissolved in 1 L water and mixed for        10-15 min until peracetic acid is generated in-situ. Both        peracetic acid and hydrogen peroxide can be tested via Lamotte        test kit code 7191-02. This solution was to be used within 6        hours to maintain a high concentration of peracetic acid.

4. Neo Pure Preparation:

Description:

-   -   Neo Pure is a powdered composition that generates peracetic acid        in-situ via TAED, sodium percarbonate and citric acid mixture.        Also, it generates hydrogen peroxide and contains a        poylglycoside wetting agent (i.e. a surfactant). Neo Pure is        equivalent to 10% peracetic acid. More particularly, the Neo        Pure had the following formulation:

Sodium percarbonate 58% w/w Tetraacetylethylenediamine (TAED) 20% w/wCitric acid (food-grade) 18% w/w Glucooon 50 G surfactant(alkylpolyglycoside)  4% w/w Total 100%

Preparation:

-   -   100 g of Neo Pure was dissolved in 1 L water and mixed for 10-15        min until peracetic acid was generated in-situ. Both peracetic        acid and hydrogen peroxide can be tested via Lamotte test kit        code 7191-02. This solution was to be used within 6 hours to        maintain a high concentration of peracetic acid.

5. Neo Pure Preparation with Alcohol:

Description:

-   -   Neo Pure is a powdered composition that generates peracetic acid        in-situ via TAED, sodium percarbonate and citric acid mixture.        Also it generates hydrogen peroxide and contains a poylglycoside        wetting agent (i.e. a surfactant). Neo Pure is equivalent to 10%        peracetic acid.

Preparation:

-   -   100 g of Neo Pure were dissolved in 1 L water and mixed for        10-15 min until peracetic acid was generated in-situ. Then, 100        ml of the solution so obtained was mixed with 100 ml ethanol 94%        for 10 minutes.

6. Liquid Peracetic Acid Preparation without Wetting Agent:

Description:

-   -   PERCID is a CFIA approved liquid preformed peracetic acid.        PERCID is a concentrated 5% peracetic acid formula composed of        mixing liquid acetic acid with liquid hydrogen peroxide.

Preparation:

-   -   200 ml of PERCID was dissolved in 1 L water and mixed for 5        minutes. A non-foaming solution free of surface-active agents        such as a wetting agent, was obtained.

7. Liquid Peracetic Acid Preparation with a Wetting Agent (i.e. aSurfactant):

Description:

-   -   PERCID is a CFIA approved liquid preformed peracetic acid.        Percid is a concentrated 5% peracetic acid formula composed of        mixing liquid acetic acid with liquid hydrogen peroxide. PERCID        solution is mixed a liquid wetting agent (i.e. surfactant) APG        325.

Preparation:

-   -   200 ml of PERCID was dissolved in 1 L water and mixed for 5        minutes. Then, 5 g of APG® 325 was added to the solution so        obtained, and mixed for 5 minutes. A foaming PAA solution was        obtained.

8. Liquid Peracetic Acid Preparation with Wetting Agent (i.e.Surfactant) and Alcohol:

Description:

-   -   PERCID is a CFIA approved liquid preformed peracetic acid.        PERCID is a concentrated 5% peracetic acid formula composed of        mixing liquid acetic acid with liquid hydrogen peroxide. PERCID        solution is mixed a liquid wetting agent (i.e. surfactant) APG®        325.

Preparation:

-   -   200 ml of PERCID was dissolved in 1 L water, and mixed for 5        minutes. Then, 5 g of APG® 325 were added to the resulting        solution, and mixed for 5 minutes. A foaming PAA solution was        obtained. Then, 100 ml of this foaming PAA was mixed with 100 ml        ethanol 94% for 10 minutes, to provide the liquid peracetic acid        preparation with wetting agent and alcohol.

9. Powdered Peracetic Acid Preparation with Alcohol:

Description:

-   -   A blend of sodium percarbonate (62%), TAED (20%) and citric acid        (18%) that generates peracetic acid and hydrogen peroxide        in-situ. It is a non-foaming solution and free of surface active        agents. This powdered PAA is equivalent to 10% peracetic acid.

Preparation:

-   -   100 g of Powdered PAA were dissolved in 1 L water, and mixed for        10-15 min until peracetic acid is generated in-situ. Then, 100        ml of the solution so obtained was mixed with 100 ml ethanol 94%        for 10 minutes.

10. Liquid Peracetic Acid Preparation with Alcohol:

Description:

-   -   PERCID is a CFIA approved liquid preformed peracetic acid.        PERCID is a concentrated 5% peracetic acid formula composed of        mixing liquid acetic acid with liquid hydrogen peroxide.

Preparation:

-   -   200 ml of PERCID were dissolved in 1 L water, and mixed for 5        minutes. A non-foaming PAA solution was obtained. Then, 100 ml        of this non-foaming PAA was mixed with 100 ml Ethanol 94% for 10        minutes, to provide the liquid peracetic acid preparation with        alcohol.

II—Preparation of Different Seeds Grains.

Seeds were mechanically cleaned and spread in stainless steelcontainers. Each 100 grams seeds were sprayed with 4 ml total solutionsdescried above via conventional trigger vaporizer. This solution isequivalent to 40 L disinfecting solution total sprayed on 1-tonne seeds.Seeds, grains and spices were selected to represent all families andtypes of seeds, grains and spices. Another criterium was to select seedsand grains contaminated with a high count of total aerobic bacteria,yeast, mold, E. coli, Salmonella sp. and other pathogenicmicroorganisms.

Seeds, grains and spices treated were:

-   -   Whole dried pea,    -   Split pea dried,    -   Pea fiber,    -   Vanilla,    -   Chia,    -   Sprouted flax and chia,    -   Flax,    -   Hemp, and    -   Black pepper seeds

TABLE 1 III- RESULTS on whole dried pea Total count Aerobic - PhysicalTreatments CFU/g bacteria Characteristics Organoleptic 0- Untreated Av =800,000 No effect No effect n1 = 700,000 n2 = 890,000 Av = 1,000 Y&M n1= 1000/n2 = 1000 1- Wetting agent APG 325 Av = 925,000 No effect Noeffect 0.5% n1 = 850,000 n2 = 1 million Av = 1,000 Y&M n1 = 1000/n2 =1000 2- Alcohol - ethanol Av = 900,000 No effect No effect 50% n1 =800,000 n2 = 1 million Y&M not detected n1, n2 not detected 3- PowderedPAA Alone Av = 300,000 No effect No effect 4 kg/40 L n1 = 250,000 n2 =350,000 Y&M not detected n1, n2 not detected 4- Neo Pure Av = 150,000 Noeffect No effect 4 kg/40 L n1 = 170,000 n2 = 130,000 Y&M not detectedn1, n2 not detected 5- Neo Pure + alcohol Av = 60,000 No effect Noeffect 2 kg/20 L + 20 L n1 = 60,000 n2 = 60,000 Y&M not detected n1, n2not detected 6- Percid -Liquid PAA alone Av = 250,000 No effect Noeffect 8 L/40 L n1 = 230,000 n2 = 270,000 Y&M not detected n1, n2 notdetected 7- Percid -Liquid PAA Av = 50,000 No effect No effect 8 L/40L + wetting agent n1 = 80,000 n2 = 20,000 Y&M not detected n1, n2 notdetected 8- Percid -Liquid PAA + Av = 5,000 No effect No effect wettingagent + alcohol (94%) n1 = 5,000 4 L/20 L + 20 L ethanol + n2 = 5,000wetting agent Y&M not detected n1, n2 not detected 9- Powdered PAA + Av= 70,000 No effect No effect alcohol (94%) n1 = 85,000 2 kg/20 L + 20 Lethanol n2 = 55,000 Y&M not detected n1, n2 not detected 10- Percid-Liquid PAA + Av = 60,000 No effect No effect alcohol n1 = 80,000 4 L/20L + 20 L ethanol n2 = 40,000 (94%) Y&M not detected n1, n2 not detected

Conclusion:

The polyglycoside wetting agent (i.e. surfactant) was not bactericidaland can act as a food source for the bacteria

Alcohol (50% concentration) applied at ratio of 40 L per 1 tonne peaswas not a strong bactericidal agent.

Peracetic acid either preformed via liquid formulations or in-situgenerated via powdered formulations, showed to be a strong bactericidalagent and reduced the level of bacteria, yeast and mold significantly.

Wetting agent (i.e. surfactant) combined to peracetic acid formulationsincreased the efficiency of the oxidizer and showed to be synergisticwith peracetic acid.

Alcohol (ethanol) combined with peracetic acid formulations increasedthe efficiency of the oxidizer and showed to be synergistic withperacetic acid.

Both alcohol and wetting agent (i.e. surfactant) increase the coverageof peracetic acid and help this limited amount of solution (40 L per 1tonne seed) to better cover the seeds and penetrate the seeds and targetmicroorganisms. They showed a synergistic effect that is higher than theone of the peracetic acid with a wetting agent or the peracetic acidwith an alcohol.

TABLE 2 IV- RESULTS on whole split pea Total count Aerobic PhysicalTreatments CFU/g bacteria Characteristics Organoleptic 0- Untreated Av =800,000 No effect No effect n1 = 750,000 n2 = 850,000 Av = 1,000 Y&M n1= 1000/n2 = 1000 1- Wetting agent APG 325 Av = 900,000 No effect Noeffect 0.5% n1 = 930,000 n2 = 870,000 Av = 1,000 Y&M n1 = 1000/n2 = 10002- Alcohol - ethanol Av = 700,000 No effect No effect 50% n1 = 700,000n2 = 700,000 Y&M not detected n1, n2 not detected 3- Powdered PAA AloneAv = 400,000 No effect No effect 4 kg/40 L n1 = 430,000 n2 = 370,000 Y&Mnot detected n1, n2 not detected 4- Neo Pure Av = 350,000 No effect Noeffect 4 kg/40 L n1 = 450,000 n2 = 250,000 Y&M not detected n1, n2 notdetected 5- Neo Pure + alcohol Av = 100,000 No effect No effect 2 kg/20L + 20 L ethanol (94%) n1 = 80,000 n2 = 120,000 Y&M not detected n1, n2not detected 6- Percid -Liquid PAA alone Av = 300,000 No effect Noeffect 8 L/40 L n1 = 400,000 n2 = 200,000 Y&M not detected n1, n2 notdetected 7- Percid -Liquid PAA Av = 200,000 No effect No effect 8 L/40L + wetting agent n1 = 200,000 n2 = 200,000 Y&M not detected n1, n2 notdetected 8- Percid -Liquid PAA + Av = 50,000 No effect No effect wettingagent + alcohol (94%) n1 = 80,000 4 L/20 L + 20 L ethanol + n2 = 30,000wetting agent Y&M not detected n1, n2 not detected 9- Powdered PAA + Av= 150,000 No effect No effect alcohol (94%) n1 = 200,000 2 kg/20 L + 20L ethanol n2 = 100,000 Y&M not detected n1, n2 not detected 10- Percid-Liquid PAA + Av = 150,000 No effect No effect alcohol 4 L/20 L + n1 =130,000 20 L ethanol (94%) n2 = 170,000 Y&M not detected n1, n2 notdetected

Conclusion:

The polyglycoside wetting agent (i.e. surfactant) was not bactericidaland can act as a food source for the bacteria

Alcohol (50% concentration) applied at ratio of 40 L per 1 tonne peaswas not a strong bactericidal agent on split pea.

Peracetic acid either preformed via liquid formulations or in-situgenerated via powdered formulations, was a strong bactericidal agent andreduced the level of bacteria, yeast and mold significantly.

Wetting agent (i.e. surfactant) combined to peracetic acid formulationsincreased the efficiency of the oxidizer and showed to be synergisticwith peracetic acid.

Alcohol (ethanol) combined to peracetic acid formulations increased theefficiency of the oxidizer and thus showed to be synergistic withperacetic acid.

Both alcohol and wetting agent (i.e. surfactant) increased the coverageof peracetic acid and helped this limited amount of solution (40 L per 1tonne seed) to better cover the seeds and penetrate the seeds and targetmicroorganisms. They showed a synergistic effect that is higher than theone of the peracetic acid with a wetting agent or the peracetic acidwith an alcohol.

TABLE 3 V- RESULTS on pea fiber Total count Aerobic Physical TreatmentsCFU/g bacteria Characteristics Organoleptic 0- Untreated Av = 900,000 Noeffect No effect n1 = 700,000 n2 = 1.1 million Av = 1,000 Y&M n1 =1000/n2 = 1000 1- Surfactant APG 325 Av = 900,000 No effect No effect0.5% n1 = 850,000 n2 = 950,000 Av = 1,000 Y&M n1 = 1000/n2 = 1000 2-Alcohol - ethanol Av = 700,000 No effect No effect 50% n1 = 500,000 n2 =900,000 Y&M not detected n1, n2 not detected 3- Powdered PAA Alone Av =500,000 No effect No effect 4 kg/40 L n1 = 700,000 n2 = 300,000 Y&M notdetected n1, n2 not detected 4- Neo Pure Av = 500,000 No effect Noeffect 4 kg/40 L n1 = 450,000 n2 = 550,000 Y&M not detected n1, n2 notdetected 5- Neo Pure + alcohol Av = 250,000 No effect No effect 2 kg/20L + 20 L ethanol (94%) n1 = 200,000 n2 = 300,000 Y&M not detected n1, n2not detected 6- Percid -Liquid PAA alone Av = 600,000 No effect Noeffect 8 L/40 L n1 = 600,000 n2 = 600,000 Y&M not detected n1, n2 notdetected 7- Percid -Liquid PAA Av = 400,000 No effect No effect 8 L/40L + surfactant n1 = 500,000 n2 = 300,000 Y&M not detected n1, n2 notdetected 8- Percid -Liquid PAA + Av = 300,000 No effect No effectsurfactant + alcohol (94%) n1 = 330,000 4 L/20 L + 20 L ethanol + n2 =270,000 surfactant Y&M not detected n1, n2 not detected 9- PowderedPAA + Av = 300,000 No effect No effect alcohol (94%) n1 = 400,000 2kg/20 L + 20 L ethanol n2 = 200,000 Y&M not detected n1, n2 not detected10- Percid -Liquid PAA + Av = 450,000 No effect No effect alcohol 4 L/20L + n1 = 600,000 20 L ethanol (94%) n2 = 300,000 Y&M not detected n1, n2not detected

Conclusion:

The solution affected the fiber pea size due to humidity. However,drying can restore the size of fiber pea as the untreated.

The polyglycoside wetting agent (i.e. surfactant) was not bactericidaland can act as a food source for the bacteria

Alcohol (50% concentration) applied at ratio of 40 L per 1 tonne peaswas not a strong bactericidal agent on pea fiber at used concentration(i.e. 40 L of alcohol 50% active per 1 tonne).

Peracetic acid either preformed via liquid formulations or in-situgenerated via powdered formulations, was a strong bactericidal agent andreduced the level of bacteria, yeast and mold significantly.

Wetting agent (i.e. surfactant) combined with peracetic acid formulationincreased the efficiency of the oxidizer and thus showed to besynergistic with peracetic acid.

Alcohol (ethanol) combined with peracetic acid formulations increasesthe efficiency of the oxidizer and thus showed to be synergistic withperacetic acid.

Both alcohol and wetting agent (i.e. surfactant) increased the coverageof peracetic acid and helped this limited amount of solution (40 L per 1tonne seed) to better cover the seeds and penetrate the seeds and targetmicroorganisms. They showed a synergistic effect that is higher that theone of the peracetic acid with a wetting agent or the peracetic acidwith an alcohol.

TABLE 4 VI- RESULTS on vanilla Total count Aerobic Physical TreatmentsCFU/g bacteria Characteristics Organoleptic 0- Untreated Av = 2 millionsNo effect No effect n1 = 1.5 millions n2 = 2.5 millions Av = 1,000 Y&Mn1 = 1000/n2 = 1000 1- Wetting agent APG 325 Av = 2 millions No effectNo effect 0.5% n1 = 1.3 millions n2 = 2.7 millions Av = 1,000 Y&M n1 =1000/n2 = 1000 2- Alcohol - ethanol Av = 1 million No effect No effect50% n1 = 1 million n2 = 1 million Av = 1,000 Y&M n1 = 1000/n2 = 1000 3-Powdered PAA Alone Av = 900,000 No effect No effect 4 kg/40 L n1 =600,000 n2 = 1.2 millions Y&M not detected n1, n2 not detected 4- NeoPure Av = 500,000 No effect No effect 4 kg/40 L n1 = 600,000 n2 =400,000 Y&M not detected n1, n2 not detected 5- Neo Pure + alcohol Av =200,000 No effect No effect 2 kg/20 L + 20 L ethanol (94%) n1 = 150,000n2 = 250,000 Y&M not detected n1, n2 not detected 6- Percid -Liquid PAAalone Av = 500,000 No effect No effect 8 L/40 L n1 = 600,000 n2 =400,000 Y&M not detected n1, n2 not detected 7- Percid -Liquid PAA Av =300,000 No effect No effect 8 L/40 L + wetting agent n1 = 320,000 n2 =280,000 Y&M not detected n1, n2 not detected 8- Percid -Liquid PAA + Av= 300,000 No effect No effect wetting agent + alcohol (94%) n1 = 310,0004 L/20 L + 20 L ethanol + n2 = 290,000 wetting agent Y&M not detectedn1, n2 not detected 9- Powdered PAA + Av = 300,000 No effect No effectalcohol (94%) n1 = 300,000 2 kg/20 L + 20 L ethanol n2 = 300,000 Y&M notdetected n1, n2 not detected 10- Percid -Liquid PAA + Av = 300,000 Noeffect No effect alcohol 4 L/20 L + n1 = 330,000 20 L ethanol (94%) n2 =270,000 Y&M not detected n1, n2 not detected

Conclusion:

The solutions were sprayed on vanilla as received in rod shape to reducethe level of total aerobic count.

The polyglycoside wetting agent (i.e. surfactant) was not bactericidaland can act as a food source for the bacteria

Alcohol (50% concentration) applied at ratio of 40 L per 1 tonne vanillawas not a strong bactericidal agent on vanilla.

Peracetic acid either preformed via liquid formulations or generatedin-situ via powdered formulations, was a strong bactericidal agent andreduced the level of bacteria, yeast and mold significantly.

Wetting agent (i.e. surfactant) combined with peracetic acidformulations increased the efficiency of the oxidizer and thus showed tobe synergistic with peracetic acid.

Alcohol (ethanol) combined with peracetic acid formulations increasedthe efficiency of the oxidizer and thus showed to be synergistic withperacetic acid.

Both alcohol and wetting agent (i.e. surfactant) increased the coverageof peracetic acid and helped this limited amount of solution (40 L per 1tonne seed) to better cover the seeds and penetrate the seeds and targetmicroorganisms. They showed a synergistic effect that is higher than theone of the peracetic acid with a wetting agent or the peracetic acidwith an alcohol.

TABLE 5 VII- RESULTS on chia seeds Total count Aerobic PhysicalTreatments CFU/g bacteria Characteristics Organoleptic 0- Untreated Av =1 million Mucilage No effect n1 = 1.1 million observed n2 = 900,000 Av =10,000 Y&M n1 = 10,000/n2 = 10,000 1- Wetting agent APG 325 Not testeddue to Mucilage Mucilage 0.5% mucilage observed observed 2- Alcohol -ethanol Av = 510,000 No mucilage No mucilage 50% n1 = 520,000 n2 =500,000 Av = 10,000 Y&M n1 = 10,000/n2 = 10,000 3- Powdered PAA AloneNot tested due to Mucilage Mucilage 4 kg/40 L mucilage observed observed4- Neo Pure Not tested due to Mucilage Mucilage 4 kg/40 L mucilageobserved observed 5- Neo Pure + alcohol Av = 300,000 No mucilage Noeffect. 2 kg/20 L + 20 L ethanol (94%) n1 = 330,000 n2 = 270,000 Av =1,000 Y&M n1 = 1,000/n2 = 1,000 6- Percid -Liquid PAA alone Not testeddue to Mucilage Mucilage 8 L/40 L mucilage observed observed 7- Percid-Liquid PAA Not tested due to Mucilage Mucilage 8 L/40 L + wetting agentmucilage observed observed 8- Percid -Liquid PAA + Av = 300,000 Nomucilage No effect. wetting agent + alcohol (94%) n1 = 350,000 4 L/20L + 20 L ethanol + n2 = 250,000 surfactant Av = 1,000 Y&M n1 = 1,000/n2= 1,000 9- Powdered PAA + Av = 400,000 No mucilage No effect. alcohol(94%) n1 = 420,000 2 kg/20 L + 20 L ethanol n2 = 380,000 Av = 1,000 Y&Mn1 = 1,000/n2 = 1,000 10- Percid -Liquid PAA + Av = 200,000 No mucilageNo effect. alcohol 4 L/20 L + n1 = 150,000 20 L ethanol (94%) n2 =250,000 Y&M not detected n1, n2 not detected

Conclusion:

The presence of alcohol prevents the release of mucilage.

The PAA in 50% Alcohol seems efficacious in reducing bacteria and yeast.

TABLE 6 VIII- RESULTS on flax seeds Total count Aerobic PhysicalTreatments CFU/g bacteria Characteristics Organoleptic 0- Untreated Av =5 millions Mucilage No effect n1 = 6.5 millions observed n2 = 3.5millions Av = 1,000 Y&M n1 = 1,000/n2 = 1,000 1- Wetting agent APG 325Not tested due to Mucilage Mucilage 0.5% mucilage observed observed 2-Alcohol - ethanol Av = 2 millions No mucilage No mucilage 50% n1 = 1.8millions n2 = 2.2 millions Av = 1,000 Y&M n1 = 1,000/n2 = 1,000 3-Powdered PAA Alone Not tested due to Mucilage Mucilage 4 kg/40 Lmucilage observed observed 4- Neo Pure Not tested due to MucilageMucilage 4 kg/40 L mucilage observed observed 5- Neo Pure + alcohol Av =1 million No mucilage No effect. 2 kg/20 L + 20 L ethanol (94%) n1 = 1million n2 = 1 million Y&M not detected n1, n2 not detected 6- Percid-Liquid PAA alone Not tested due to Mucilage Mucilage 8 L/40 L mucilageobserved observed 7- Percid -Liquid PAA Not tested due to MucilageMucilage 8 L/40 L + wetting agentt mucilage observed observed 8- Percid-Liquid PAA + Av = 700,000 No mucilage No effect. wetting agent +alcohol (94%) n1 = 850,000 4 L/20 L + 20 L ethanol + n2 = 550,000surfactant Y&M not detected n1, n2 not detected 9- Powdered PAA + Av =900,000 No mucilage No effect. alcohol (94%) n1 = 915,000 2 kg/20 L + 20L ethanol n2 = 885,000 Y&M not detected n1, n2 not detected 10- Percid-Liquid PAA + Av = 800,000 No mucilage No effect. alcohol 4 L/20 L + n1= 800,000 20 L ethanol (94%) n2 = 800,000 Y&M not detected n1, n2 notdetected

Conclusion:

The presence of alcohol prevented the release of mucilage.

Macroscopically, mucilage was not observed on seeds treated withalcohol.

The PAA in 50% alcohol was efficacious in reducing bacteria and yeast.

TABLE 7 IX- RESULTS on sprouted flax and chia Total count AerobicPhysical Treatments CFU/g bacteria Characteristics Organoleptic 0-Untreated Av = 2 millions Mucilage No effect n1 = 2.5 millions observed,n2 = 1.5 millions very wet Av = 1,000 Y&M n1 = 1,000/n2 = 1,000 1-Wetting agent APG 325 Not tested due to Mucilage Mucilage 0.5% mucilageobserved observed 2- Alcohol - ethanol Av = 600,000 No mucilage Nomucilage 50% n1 = 550,000 n2 = 650,000 Av = 1,000 Y&M n1 = 1,000/n2 =1,000 3- Powdered PAA Alone Not tested due to Mucilage Mucilage 4 kg/40L mucilage observed observed 4- Neo Pure Not tested due to MucilageMucilage 4 kg/40 L mucilage observed observed 5- Neo Pure + alcohol Av =200,000 No mucilage No effect. 2 kg/20 L + 20 L ethanol (94%) n1 =220,000 n2 = 180,000 Y&M not detected n1, n2 not detected 6- Percid-Liquid PAA alone Not tested due to Mucilage Mucilage 8 L/40 L mucilageobserved observed 7- Percid -Liquid PAA Not tested due to MucilageMucilage 8 L/40 L + wetting agent mucilage observed observed 8- Percid-Liquid PAA + Av = 500,000 No mucilage No effect. wetting agent +alcohol (94%) n1 = 525,000 4 L/20 L + 20 L ethanol + n2 = 475,000wetting agent Y&M not detected n1, n2 not detected 9- Powdered PAA + Av= 600,000 No mucilage No effect. alcohol (94%) n1 = 600,000 2 kg/20 L +20 L ethanol n2 = 600,000 Y&M not detected n1, n2 not detected 10-Percid -Liquid PAA + Av = 600,000 No mucilage No effect. alcohol 4 L/20L + n1 = 500,000 20 L ethanol (94%) n2 = 700,000 Y&M not detected n1, n2not detected

Conclusion:

Humidity including alcohol solution may affect the sprouted flax andchia. It should be dried well.

The presence of alcohol was shown to prevent the release of mucilage

Alcohol was shown to act as a bactericidal agent but not very strong.

The PAA in 50% alcohol was shown to be efficacious in reducing bacteriaand yeast.

Macroscopically, mucilage was not observed on seeds treated withalcohol.

TABLE 8 X- RESULTS on hemp Total count Aerobic Physical Treatments CFU/gbacteria Characteristics Organoleptic 0- Untreated Av = 2 millions Noeffect No effect n1 = 1.7 millions n2 = 2.3 millions Av = 1,000 Y&M n1 =1000/n2 = 1000 1- Wetting agent APG 325 Av = 2 millions No effect Noeffect 0.5% n1 = 1.5 millions n2 = 2.5 millions Av = 1,000 Y&M n1 =1000/n2 = 1000 2- Alcohol - ethanol Av = 1 million No effect No effect50% n1 = 800,000 n2 = 1.2 millions Av = 1,000 Y&M n1 = 1000/n2 = 1000 3-Powdered PAA Alone Av = 500,000 No effect No effect 4 kg/40 L n1 =600,000 n2 = 400,000 Y&M not detected n1, n2 not detected 4- Neo Pure Av= 300,000 No effect No effect 4 kg/40 L n1 = 300,000 n2 = 300,000 Y&Mnot detected n1, n2 not detected 5- Neo Pure + alcohol Av = 100,000 Noeffect No effect 2 kg/20 L + 20 L ethanol (94%) n1 = 120,000 n2 = 80,000Y&M not detected n1, n2 not detected 6- Percid -Liquid PAA alone Av =310,000 No effect No effect 8 L/40 L n1 = 300,000 n2 = 320,000 Y&M notdetected n1, n2 not detected 7- Percid -Liquid PAA Av = 300,000 Noeffect No effect 8 L/40 L + wetting agent n1 = 320,000 n2 = 280,000 Y&Mnot detected n1, n2 not detected 8- Percid -Liquid PAA + Av = 200,000 Noeffect No effect wetting agent + alcohol (94%) n1 = 100,000 4 L/20 L +20 L ethanol + n2 = 300,000 surfactant Y&M not detected n1, n2 notdetected 9- Powdered PAA + Av = 200,000 No effect No effect alcohol(94%) n1 = 200,000 2 kg/20 L + 20 L ethanol n2 = 200,000 Y&M notdetected n1, n2 not detected 10- Percid -Liquid PAA + Av = 300,000 Noeffect No effect alcohol 4 L/20 L + n1 = 330,000 20 L ethanol (94%) n2 =270,000 Y&M not detected n1, n2 not detected

Conclusion:

The above-mentioned solutions were sprayed on hemp seeds to achieve areduction of the level of total aerobic count.

The polyglycoside wetting agent (i.e. surfactant) was not bactericidal.

Alcohol (50% concentration) applied at ratio of 40 L per 1 tonne hempseed was not a strong bactericidal agent on hemp.

Peracetic acid either preformed via liquid formulations or generatedin-situ via powdered formulations, was a strong bactericidal agent andreduced the level of bacteria, yeast and mold significantly.

Wetting agent (i.e. surfactant) combined with peracetic acidformulations increased the efficiency of the oxidizer and thus showed tobe synergistic with peracetic acid.

Alcohol (ethanol) combined with peracetic acid formulations increasedthe efficiency of the oxidizer and thus showed to be synergistic withperacetic acid.

Both alcohol and surfactant increase the coverage of peracetic acid andhelp this limited amount of solution (40 L per 1 tonne seed) to bettercover the seeds and penetrate the seeds and target microorganisms. Theyshowed a synergistic effect that is higher than that of the peraceticacid with a wetting agent or the peracetic acid with an alcohol.

TABLE 9 XI- Results on black pepper Total count Aerobic PhysicalTreatments CFU/g bacteria Characteristics Organoleptic 0- Untreated Av =1.5 millions No effect No effect Av = 3,000 Y&M 1- Surfactant APG 325 Av= 1.7 millions No effect No effect 0.5% Av = 3,000 Y&M 2- Alcohol -ethanol Av = 1 million No effect No effect 50% Av = 1,000 Y&M 3-Powdered PAA Alone Av = 500,000 No effect No effect 4 kg/40 L Av = 100Y&M 4- Neo Pure Av = 480,000 No effect No effect 4 kg/40 L Av = 100 Y&M5- Neo Pure + alcohol Av = 370,000 No effect No effect 2 kg/20 L + 20 Lethanol (94%) Av = 100 Y&M 6- Percid -Liquid PAA alone Av = 460,000 Noeffect No effect 8 L/40 L Y&M not detected 7- Percid -Liquid PAA Av =400,000 No effect No effect 8 L/40 L + surfactant Y&M not detected 8-Percid -Liquid PAA + Av = 380,000 No effect No effect surfactant +alcohol (94%) Y&M not detected 4 L/20 L + 20 L ethanol + surfactant 9-Powdered PAA + Av = 400,000 No effect No effect alcohol (94%) Y&M notdetected 2 kg/20 L + 20 L ethanol 10- Percid -Liquid PAA + Av = 380,000No effect No effect alcohol 4 L/20 L + Y&M not detected 20 L ethanol(94%)

Conclusion:

The disinfecting solutions sprayed on black pepper seeds can reduce thelevel of total aerobic count.

The polyglycoside surfactant is not bactericidal.

Alcohol (50% concentration) applied at ratio of 40 L per 1 tonne blackpepper seed is not a strong bactericidal agent on hemp.

Peracetic acid either preformed via liquid formulations or generatedin-situ via powdered formulations, is a strong bactericidal agent andreduces the level of bacteria, yeast and mold significantly.

Alcohol (ethanol) combined with peracetic acid formulations increasesthe efficiency of the oxidizer and thus is synergistic with peraceticacid.

Both alcohol and surfactant increase the coverage of peracetic acid andhelp this limited amount of solution (40 L per 1 tonne seed) to bettercover the seeds and penetrate the seeds and target microorganisms.

Example 2: A Trial Assessing the Efficacy of Powdered Formula (PowderedPAA with a Wetting Agent) in the Surface Disinfection of Hemp Seeds in aGrain Conditioning Facility

Protocol:

Several tonnes of hemp seeds were cleaned mechanically using regulargrain conditioning equipment. The total bacterial count was determinedto be about 18 million CFU/g (before mechanical cleaning andseparation). After mechanical cleaning, the total bacterial count wasfound to be about 2 million CFU/g. This microbial load does not complywith the market standard which is 1 million CFU/g.

Treatment with Peracetic Acid and Hydrogen Peroxide without an Alcoholand/or a Wetting Agent:

Hemp seeds (with a microbial load of about 2 million CFU/g) weresanitized with a powdered product based on sodium percarbonate, TAED andcitric acid that generates peracetic acid and hydrogen peroxide in situ.An equivalent of 4 kg of this formula were dissolved in potable waterand mixed thoroughly for 10 minutes and then applied to 1 tonne of hempseeds and allowed to remain in contact with them for 30 minutes. Theseeds were thoroughly dried after the treatment. The results did notshow a significant reduction in microbial load as compared to untreatedseeds (2 million CFU/g). These results were not satisfactory. Inaddition, coliforms, E. coli, yeast and mold were detected. The powderedformulation that generates PAA in-situ was based on 70% w/w sodiumpercarbonate, mixed with 20% w/w TAED and mixed with 10% citric acid.

Treatment with Formula (Peracetic Acid Generated In-Situ with a WettingAgent):

1 tonne of cleaned hemp seeds (2 million CFU per gram) were sanitizedwith a 0.4% concentration (4 kg of formula 18/18). Said formula 18/18 ispowdered formulation is based on 40% sodium percarbonate, mixed with 20%TAED; mixed with 18% potassium silicate; mixed with 18% EDTA acid; andfinally mixed with 4% Bioterge AS 90 surfactant. The 4 kg were dilutedin 40 L water and were mixed thoroughly for 10 minutes and then appliedto treat 1 tonne of hemp seed for 30 minutes, then the treated seedswere dried very well as per the grain conditioner process. The resultsshowed a reduction in total bacterial count to 54,000 CFU per gram.These results were satisfactory and complied with the market standards.Coliforms, E. coli, yeast and mold were not detected.

Conclusions:

Based on the results shown above, there was noted a synergy betweenoxidizers (i.e. peracetic acid and hydrogen peroxide) and wetting agent(i.e. surfactant) in reducing the populations of human pathogens onedible seeds.

Example 3: Determination of Contact-Time Efficacy of Powdered PAACombined with Alcohol and a Wetting Agent

Objective:

The objective of this study is to determine the effective contact-timeof the sanitizing solutions (PAA with an alcohol and a wetting agent)sprayed on hemp seeds in controlling pathogens.

Protocol:

1 kg of hemp seeds per mix was treated with 50 ml of solution byapplying small amounts at a time using a hand sprayer and mixingthoroughly in between. Batches were stored in 3.3 L containers at roomtemperature with lids on to avoid loss of moisture due to evaporation.

Solutions Used:

-   -   Neo-Pure (5%), (50 g Neo Pure dissolved in 1 L water and mixed        for 15 minutes)    -   Neo-Pure/Ethanol (5% Neo Pure dissolved in 50% water/50%        alcohol),    -   Mock (H₂O)

Samples were taken at the indicated time points and plated immediatelywith the exception of the +1 hr time point in the experiment of 8/25(This sample was taken at +1 hr but stored at 4° C. o/n and plated thenext morning).

Results Batch Treatment 2014-08-25 Triplicates

TABLE 10 +1 hr +21 hrs +48 hrs +72 hrs UTC 786,000 3,200,000 620,000890,000 mock 701,000 793,000 400,000 765,000 Neo-Pure 1,000,000 195,000272,000 101,000 Neo-Pure EtOH 182,000 23,000 22,000 73,000

UTC means untreated and aforesaid data were reported on FIGS. 1 to 3.

Batch Treatment 2014-09-10 Pentuplicates

TABLE 11 starting point +3 hr o/n +24 hrs +48 hrs UTC 12,700,000 Neo-17,700,000 8,400,000 2,660,000 4,520,000 Pure Neo- 2,400,000 1,600,0001,600,000 1,480,000 Pure EtOH

UTC means untreated and aforesaid data were reported on FIGS. 4 to 6.

Conclusions:

Under lab conditions (room temperature: 20° C. to 27° C.) the efficacyof both Neo-Pure only and Neo-Pure+EtOH increased significantly withlonger incubation times (≧24 hours)

This effect was much more prominent at the beginning of the treatmentwith Neo-Pure only. However, it was noted that no significant regrowthof bacteria was observed within the first 48 hours post treatment ifEtOH was present.

Once seeds were treated with PAA+alcohol and/or wetting agent, thesanitizing solution continues to work for hours and reduce thepopulation of bacteria. However, after 48 hrs, seeds had to be dried toreduce the moisture content below 10% in order to prevent regrowth ofmicroorganisms. A moisture content below 10% is a usual standard of theindustry to prevent a growth of microorganisms.

Chia Seed Treatment in Small Batches with 50% Ethanol

1 kg aliquots of chia seeds were weighted into clean containerssterilized with 70% ethanol. Neo-Pure solution was prepared bydissolving Neo-Pure powder (a powdered peracetic acid precursoravailable from Agri-Neo) in tap water to a final concentration of 10%(w/v) and incubated at room temperature (RT) for 15 minutes to allow theformation of active peracetic acid (PAA) as the active ingredient.Subsequently, ethanol was added to a final concentration of 50% (v/v) togenerate sanitizing solution containing 5% Neo-Pure (w/v) and 50%ethanol (v/v). 50 ml solution (target rate: 50 l/tonne) was applied tothe seeds under vigorous mixing using a small hand vaporizer; PAAactivity was confirmed to be >160 ppm using test strips (LaMotteInsta-Test Analytic Peracetic Acid) during application. Post treatment,all aliquots were stored at RT in sealed containers until sampling.Samples were taken 12 and 24 hours hours post treatment and plated on 3MPetrislides within 2 hours of sampling to determine total aerobiccounts.

Sample processing was conducted according to manufacturersrecommendations; in brief, 7 g sample were added to 700 ml water andhomogenized for 2 min using a handmixer. A dilution series was generatedusing sterile 9 ml buffered peptone water aliquots (3M) and 1 ml of therelevant dilutions were plated onto 3M Petrifilm Aerobic Count Plates.Petrifilms were incubated at 31° C. for 72 hours before counting.

The results of these trials are summarized in the table below.

TABLE 12 Treated 50 l/tonne Treated 100 l/tonne Incubated IncubatedIncubated Incubated Untreated 12 hrs 24 hrs 12 hrs 24 hrs Total   4 ×10⁶ 1.6 × 10⁶ 3.8 × 10⁵ 8.8 × 10⁵ 3.2 × 10⁵ Aerobic Count (CFU/g)Coliforms 1.5 × 10⁴ 1.4 × 10³ 600 1.4 × 10³ 400 (CFU/g)

As indicated in the table above, at 5% Neo-Pure, 50% ethanol, chia seedswere successfully sanitized. Extending contact time up to 24 hrsimproved efficacy. There was no significant benefit demonstrated toapplying the treatment solution at a rate higher than 50 l/tonne.Mucilage release was completely suppressed.

Treatment of Mucilaginous Seeds with Alcohol at 20% and LowerConcentrations

Tests were conducted to determine (a) whether a 20% ethanol formulation,which is combustible but not flammable, would effectively sanitize seedsand (b) the minimum ethanol concentration required to prevent mucilagerelease.

A treatment solution was generated composed of (a) water: balance up to100% (v/v), (b) Neo-Pure: 10% (v/v), and (c) Active Ethanol: 20% (v/v).A minimum of 4 ml of the treatment solution was sprayed, using ahandheld atomizing sprayer while mixing, on 100 gram samples of chia andflax seeds. The samples were allowed to air dry and checked for formucilage. No mucilage was observed for either the flax or chia seeds.This same experiment was repeated with 19%, 18%, 17% and 16% ethanol inthe treatment solution with no mucilage observed. The experiment wasrepeated again with 15% ethanol in the treatment solution and aboutaround 5% of the seeds (both flax and chia) released mucilage. Theexperiment was repeated again with 14% ethanol in the treatment solutionand significant amounts of mucilage production were observed.

Another treatment solution was made up composed of (a) water: balance upto 100% (v/v), (b) Neo-Pure: 10% (v/v), (c) Active Ethanol: 10% (v/v)and (d) Propylene Glycol 10% (v/v). A minimum of 4 ml of the treatmentsolution was sprayed, using a handheld atomizing sprayer while mixing,on 100-gram samples of chia and flax seeds. The samples were allowed toair dry and checked for mucilage. No mucilage was observed for eitherthe flax or chia seeds. This same experiment was repeated with 10%ethanol and 5% propylene glycol with no mucilage observed. However, infurther experiments with 10% ethanol and less than 5% propylene glycol,some mucilage was observed.

Treatment of Various Seeds with 20% Solvent Treatment Solutions

150 g aliquots of various types of seeds were weighted into cleancontainers sterilized with 70% ethanol to generate treated and untreatedcontrol samples. 10 ml liquid Neo-Pure was mixed with 70 ml water and 20ml ethanol added to obtain 100 ml of a treatment mixture containing 10%Neo-Pure (0.5% peracetic acid (PAA) w/v final concentration activeingredient) and 20% v/v ethanol. 7.5 ml solution (target rate: 50l/tonne) were applied to the seeds under vigorous mixing using a smallhand vaporizer; PAA activity was confirmed to be >160 ppm using teststrips (LaMotte Insta-Test Analytic Peracetic Acid) during application.Post spraying, the final application rate was confirmed by determiningweight added to the sample.

Seeds were stored at room temperature over night. After approximately 16hours, 3 independent 10 g aliquots were removed from treated anduntreated seed pools and processed to be analyzed on 3M Petrifilm slidesto determine total aerobic counts. Sample processing was conductedaccording to manufacturers recommendations; in brief, 10 g sample and 90ml sterile buffered peptone water (3M) were transferred into a sterileFBAG-04 filter blender bag and processed in a stomacher at 300 rpm for 1min. A dilution series was generated using sterile 9 ml buffered peptonewater aliquots (3M) and 1 ml of the relevant dilutions were plated onto3M Petrifilm Aerobic Count Plates. Petrifilms were incubated at 31° C.for 72 hours before counting.

Results for sprouted flax seed are summarized in the table below.

TABLE 13 Total Aerobic Count Standard Sample (CFU/g) Deviation P-ValueSprouted flax, 268 × 10⁶ 6 × 10⁶ 0.0000003 untreated control Sproutedflax,  15 × 10⁶ 2 × 10⁶ treated at 50 l/tonne

Results for sprouted white quinoa seeds are summarized in the tablebelow.

TABLE 14 Total Aerobic Count Standard Sample (CFU/g) Deviation P-ValueSprouted white quinoa, 265 × 10⁶ 20 × 10⁶ 0.0000239 untreated controlSprouted white quinoa,  5 × 10⁶  2 × 10⁶ treated at 56 l/tonne

Results for sprouted white millet seeds are summarized in the tablebelow.

TABLE 15 Total Aerobic Count Standard Sample (CFU/g) Deviation P-ValueSprouted millet, 7 × 10⁵ 8 × 10⁵ 0.194 untreated control Sproutedmillet, 3 × 10³ 5 × 10³ treated at 53 l/tonne

Results for sprouted amaranth seeds are summarized in the table below.

TABLE 16 Total Aerobic Count Standard Sample (CFU/g) Deviation P-ValueSprouted amaranth, 4.8 × 10⁶ 9.6 × 10⁴ 0.001 untreated control Sproutedamaranth,  <1 × 10⁴ 0 treated at 53 l/tonne

Results for a blend of sprouted seeds including flax and chia aresummarized in the table below.

TABLE 17 Total Aerobic Count Standard Sample (CFU/g) Deviation P-ValueSprouted seed blend, 14 × 10⁶ 2.8 × 10⁶ 0.000004 untreated controlSprouted seed blend,  3 × 10⁵  1 × 10⁵ treated at 44 l/tonne

Treatment of the seeds described above with a Neo-Pure solutioncontaining 20% ethanol reduced total aerobic counts on these seeds by95%-99.5% with no negative effect on seed appearance and no detectablerelease of mucilage.

Initial Application Method

Large scale production runs were simulated in a lab using a small hopperand auger setup to treat larger amount of seeds in a semi-continuoussystem. Hemp or Chia seeds were released from a reservoir and fell intoa hopper. The hopper drained into an auger at the bottom of the hopper.A treatment solution containing 5% Neo-Pure powder (w/v) and 50% ethanol(v/v) was applied to the seeds as they fell into the hopper by a nozzlearray consisting of three 8001vs nozzles operating at 30 psi (see FIG.7). Further mixing of the treatment solution and the seeds occurred asthe seeds travelled through the auger.

The nozzles were adjusted to generate a target delivery rate buttreatment solution volumes before and after the runs (leftover) weremeasured to determine actual application rates. The runs described belowwere not performed in parallel but at different dates.

Samples were either taken immediately (hemp) or 24 hours (chia) aftertreatment and plated on 3M Petrislides within 2 hours of sampling todetermine total aerobic microorganism counts. Sample processing wasconducted according to manufacturers recommendations. In brief, a 7 gsample was added to 700 ml water and homogenized for 2 min using ahandmixer. A dilution series was generated using sterile 9 ml bufferedpeptone water aliquots (3M) and 1 ml of the relevant dilutions wereplated onto 3M Petrifilm Aerobic Count Plates. Petrifilms were incubatedat 31° C. for 72 hours before counting.

For the hemp seed samples, processed and plated immediately aftertreatment, the lack of a post-treatment incubation period is expected tohave lowered the apparent efficacy of the treatment based on otherresults presented further above. Two trials with hemp seeds wereconducted using identical settings but for different treatment solutionapplication rates. 57 l/tonne was applied in the first trial and 113l/tonne was applied in the second run. The total aerobic counts measuredare presented in the table below.

TABLE 18 ACC untreated, ACC treated 57 ACC treated 113 Seed Type (CFU/g)l/tonne (CFU/g) l/tonne (CFU/g) Hemp 7.5 × 10⁶ 4.6 × 10⁵ 2.9 × 10⁵

As indicated in the table above, aerobic count was reduced 94% in thefirst trial and 96% in the second trial. This efficacy would likely havebeen higher if an incubation period (for example 24 hours) had beenapplied.

For the chia seed trial, the seeds were incubated over night aftertreatment. The treatment solution application rate was 120 l/tonne.Aerobic count results are presented in the table below.

TABLE 19 ACC untreated, ACC treated 120 Seed Type (CFU/g) l/tonne,incubated (CFU/g) p-value Chia 4.5 × 10⁵ 1.4 × 10⁶ 0.12

No significant reduction in microbial load was observed after treatmentof the chia seeds even though the application rate was higher than forthe hemp seeds. Numerically, ACC counts were higher after treatment andincubation than in the untreated sample, but the numerical differencewas not statistically significant.

Both seed types can be efficiently sanitized with Neo-Pure in smallscale batch experiments and so the chemistry of the treatment solutionis not believed to be the underlying cause of this difference. The mostprominent difference between hemp and chia seeds is their capacity toproduce mucilage, chia being the more mucilaginous seed. Withoutintending to be limited by theory, the inventors believe that thecombination of a mucilaginous seed with a sprayed application of thetreatment solution is responsible for the lack of activity in the chiatrial described above. Mucilage in the seed coat may be able toinactivate, absorb or consume peractic acid. In the small scale batchexperiments, the treatment solution was applied to the seeds through anatomizing sprayer. The atomizing sprayer increases the initial contactsurface, or the initial dispersion of treatment solution across theseed, relative to the nozzles used over the hopper, which delivered agenerally continuous stream of treatment solution. The improved initialdispersion of the treatment solution with an atomizing sprayer may allowthe treatment solution to neutralize more microbes before it becomesdeactivated. With the sprayer, hopper and auger set-up, the initial seedcontact surface is more limited and more nearly complete dispersion ofthe treatment solution may not occur until the seeds are mixed in theauger. Although secondary distribution occurs in the auger withinseconds of the initial application, in the case of mucilaginous seeds asignificant part of the treatment solution activity may be compromisedby mucilage if the treatment solution is not well dispersed around theseed on initial contact. Hemp, in contrast, does not produce mucilage,and is adequately treated even if the initial application of thetreatment solution is more concentrated. Consequently, secondarydistribution of treatment solution is believed to be sufficient for thetreatment of non-mucilaginous seeds.

To investigate the issues described above, and to develop a large scaletreatment process suitable for mucilaginous seeds, further trials withchia seeds were performed using a commercial seed treater (USCContinuous Treating System, USC LLC, KS). Seed treaters are typicallyused to apply small volumes of antimicrobial or antifungal agents toseeds before they are planted. In the seed treater tested, seeds fallfrom a hopper onto a seed wheel. The seed wheel scatters the seeds, andthe seeds then fall through an atomizing chamber. In the atomizingchamber, an atomizing sprayer spins while producing a mist of theapplied agent. The overall effect is that the seeds are separated fromeach other and fall through a mist of the applied agent which provides awell dispersed initial application of the agent. The seeds fall into ahorizontal drum with paddles installed along the length of the drum in astaggered pattern, which provides some secondary mixing.

In an exemplary trial, the seed treater was used to apply a treatmentsolution as discussed above to 750 ponds of chia seeds at an applicationrate of 50 l/tonne. The treated seeds were incubated overnight andsamples were plated the next day. Count results are presented in thetable below.

TABLE 20 Aerobic Yeast Mold Coliforms count count count count (CFU/g)(CFU/g) (CFU/g) (CFU/g) Chia Untreated None 704 190 35 detected TreatedNone None None None detected detected detected detected

While these chia seeds were relatively clean to start with, asignificant reduction was observed for yeast, mold and Coliforms. Allthree types of microbes were reduced to below the detection thresholdafter treatment, confirming that a mist or atomized initial applicationof the treatment solution is effective to sanitize mucilaginous seeds ina large scale process.

Reaction Time

1 kg aliquots of cleaned, unprocessed hemp seeds were weighted intoclean containers sterilized with 70% ethanol. Neo-Pure solution wasprepared by dissolving Neo-Pure powder in tap water to a finalconcentration of 10% (w/v) and incubated at room temperature (RT) for 15minutes to allow the formation of active peracetic acid (PAA) as theactive ingredient. For some trials, ethanol was added to a finalconcentration of 50% (v/v) to generate a sanitizing solution containing5% Neo-Pure (w/v) and 50% ethanol (v/v). 50 ml solution was applied tothe seeds at a target application rate of 50 l/tonne with a smallhand-held vaporizer. PAA activity was confirmed to be >160 ppm usingtest strips (LaMotte Insta-Test Analytic Peracetic Acid) duringapplication. Post treatment, all aliquots were stored at roomtemperature in sealed containers until sampling.

Samples were taken 3 hours, 21 hours, 27 hours and 48 hours posttreatment and plated on 3M Petrislides within 2 hours of sampling todetermine total aerobic counts. Sample processing was conductedaccording to manufacturers recommendations. In brief, 7 g sample wereadded to 700 ml water and homogenized for 2 min using a handmixer. Adilution series was generated using sterile 9 ml buffered peptone wateraliquots (3M) and 1 ml of the relevant dilutions were plated onto 3MPetrifilm Aerobic Count Plates. Petrifilms were incubated at 31° C. for72 hours before counting. The total aerobic count (ACC) results for thesamples are presented in FIG. 8. The UTC result is for untreated seed.“o/n” indicates samples taken at +21 hours, +24 indicates samples takenat 27 hours and +48 indicates samples taken at +48 hours.

As indicated in FIG. 8, contact times longer than 3 hours have abeneficial effect on efficacy. However, there is an upper limit to thebeneficial effect of longer contact time. With overly long contacttimes, microbial regrowth can occur. As described elsewhere in thispatent, the contact time and threat of microbial regrowth are terminatedby drying the treated seeds. The presence of ethanol not only enhancedefficacy of the treatment but also extended the time window whereeffective sanitization without microbial regrowth occurs.

The inventors believe that, under at least some circumstances, contacttime could be increased beyond 48 hours, for example to 72 hours ormore, without resulting in unacceptable microbial regrowth. However,since there was an indication that regrowth was starting in thenon-ethanol formulation in FIG. 8 at 48 hours, and there was no trendtowards increased efficacy with contact time past 27 hours, it would bepreferable to restrict incubation time to 48 hours or less, or morepreferably to 27 or 24 hours or less. In other trials described herein,adequate sanitizing effect was observed in at least some trialsessentially on initial contact, where the incubation time might havebeen only in the range of 2-20 minutes. However, efficacy appears toincrease with 3 hours or more of contact time and to an even greaterextend with 21 hours or more of contact time. As described in otherresults presented further above, contact times of 12 and 24 hours weresuccessfully used to sanitize seeds. Overall, a contact time of about 24hours provides good results over a wide range of treatment solutions andseeds. A contact time range of 16-32 hours, or 21-27 hours, is expectedto also provide good results while accommodating an ordinary workschedule. For example, the treatment solution is applied to the seedsand the seeds are transferred to holding containers on one day, and theseeds are removed from their holding containers and moved to a drier onthe following day.

The examples and embodiments described herein are for illustrativepurposes to help provide an enabling description of the invention.Various modifications or changes in light thereof will be suggested topersons skilled in the art.

1. A method for sanitizing edible seeds comprising the steps of,providing a sanitizing composition comprising water, at least onebiocidal agent and at least one alcohol in an amount (a) effective tosuppress the release of mucilage from mucilaginous seeds or (b)providing at least 15% by volume of the composition; applying thesanitizing composition to the seeds; and, drying the seeds.
 2. Themethod of claim 1 wherein the sanitizing composition is applied tomucilaginous seeds as a mist.
 3. The method of claim 1, wherein theseeds are dried at least 5 minutes, but not more than 24 hours, afterapplying the sanitizing composition.
 4. The method of claim 1 whereinthe sanitizing composition comprises at least 13% by volume of propyleneglycol or at least 15% by volume of ethanol or a mixture of ethanol andpropylene glycol comprising at a minimum concentration obtained bylinear interpolation of the values for ethanol and propylene glycol. 5.The method of claim 1 wherein the at least one alcohol is ethanol in aconcentration of no more than 20% by volume.
 6. The method of claim 1wherein the applying step is followed by mixing the seeds.
 7. The methodof claim 1 wherein the seeds are not rinsed before drying the seeds. 8.The method of claim 1 wherein the sanitizing composition is applied atno more than 15% by weight of the seeds.
 9. The method of claim 1wherein the drying step passing air through a bed of the seeds.
 10. Themethod of claim 1 wherein in the drying step the seeds are dried to awater content of 10% or less relative to the weight of the seeds or to awater content within 1% by weight of the seeds of the water content ofthe seeds before applying the sanitizing composition to the seeds. 11.The method of claim 1 wherein the sanitizing composition comprisesperacetic acid and ethanol, propylene glycol, or both.
 12. A sanitizingcomposition comprising, water; at least one biocidal agent; and, atleast one alcohol in an amount effective to suppress the release ofmucilage from mucilaginous seeds.
 13. The sanitizing composition ofclaim 12 comprising at least 15% by volume of the at least one alcohol.14. The sanitizing composition of claim 12 wherein the biocidal agent isan oxidizer, preferably peracetic acid.
 15. The sanitizing compositionof claim 12 comprising at least 13% by volume of propylene glycol or atleast 15% by volume of ethanol or a mixture of ethanol and propyleneglycol comprising at a minimum concentration obtained by linearinterpolation of the values for ethanol and propylene glycol.